Reprogramming of fibroblast cells to totipotent state by DNA demethylation

Many attempts have been made to induce high-quality embryonic stem cells such as pluripotent stem cells and totipotent stem cells, but challenges remain to be overcome such as appropriate methods and sources. Demethylation of the genome after fertilization is an important step to initiate zygote gene activation, which can lead to the development of new embryos. Here, we tried to induce totipotent stem cells by mimicking DNA demethylation patterns of the embryo. Our data showed, after induction of DNA demethylation via chemicals or knockdown of Dnmts, cells positive for Nanog, and Cdx2 emerged. These cells could differentiate into the pluripotent and trophoblast lineage cells in-vitro. After transferring these cells to the uterus, they can implant and form embryo-like structures. Our study showed the importance of DNA demethylation roles in totipotent stem cell induction and a new and easy way to induce this cell type.

www.nature.com/scientificreports/ embryogenesis-related genes after DNA demethylation 30 , and many studies show the administration of demethylation agents (mostly 5-Azacitidine (Aza)) leads to induction of stem cells, especially ICM and trophoblast stem cells [30][31][32][33][34][35] might be evidence to show the role of global DNA demethylation in the induction of totipotent stem cells. In addition, it has been shown that DNMTs could inhibit ZSCAN4 36,37 , a co-effector of DUX and totipotent stem cells 38,39 . DNMTs also regulate TERT and telomeres 40 which show a significant role of Dnmts in the totipotent state. Based on these notions, we tried to induce totipotent stem cells from fibroblast cells. Our data showed that we reprogram Nanog and Cdx2 positive cells by genome demethylation. Transferring these induced totipotent (iTot) cells directly to the uterus of pseudopregnant mice. After investigation, the development of these cells into embryo-like structures was detected.

Results
Aza could induce Nanog and Cdx2 positive cells which expressed MERVL. Fibroblast cells were chosen due to their transcription network and easy handling. Cells have been treated with different doses of Aza to identify the best dose (Fig. 1A,B). Administration of one dose of Aza in any concentration cannot lead to a high number of alkaline phosphatase-positive cells, which is a marker of embryonic stem cell proliferation. However, after treating cells with one and a half micrograms per milliliter of Aza for three days, results in a high number of alkaline-positive cells are identified (Fig. 1B). We use the differential affinity of induced cells versus fibroblast to sort the induced cells as this method is done in the isolation of embryonic stem cells from MEFs. Briefly, after trypsinization, the cells are transferred to new dishes. Fibroblast cells have a stronger affinity for culture dishes and attach sooner than induced cells. Therefore, transferring media containing iTot cells after the attachment of fibroblast can lead to cell sorting. All of the further experiments from here are carried out on sorted cells. After treating cells with Aza, some structures can be seen in isolated cells. There were small, large, and large cells with cavities that could be seen in isolated cells (Fig. 1C). Afterward, the presence of ICM marker Nanog and trophectoderm (TE) marker Cdx2 are examined by immunofluorescence. These markers have an important role in early embryonic development and embryogenesis and are considered 4C cell markers. We also checked the MERVL expression in cells. Isolated cells were positive for Cdx2 and Nanog, and expression of MERVL (as a marker of totipotent stem cells) has increased (Fig. 1D,F). The measurement of DNA methylation of these cells shows increased demethylation of the genome, which is the main alteration in this study for the induction of these structures ( Fig. 2A). It also has been shown that administration of Aza could result in the down-regulation of trophoblast and pluripotency genes in treated cells which results in overexpression of these genes 33,34 . These data are consistent with the genome demethylation of totipotent stem cells and Aza-treated cells. Interestingly, after passaging cells, we do not recognize any aging-related signs, despite the differentiation of these cells in the cell culture dish. Due to this observation, the telomere length of treated cells on different days is measured and www.nature.com/scientificreports/ it keeps increasing on the sixth day ( Fig. 2B). Demethylation can disrupt imprinting patterns. The upregulation of imprinting modulators or unchanged expression of imprinting genes can be a demonstration of methylation pattern status as well as a sign of totipotency which has been reported previously (12). Dnmt3l, Dppa3, Meg3, and Igf2 genes are selected for this reason. The results of Meg3, Dnmt3l, and Dppa3 genes which also have higher expression iTot cells versus other groups (Fig. 2C). Upregulation of these genes indicates a specific effect of genome demethylation. Indeed, expression of the Igf2 gene in imprinting loci is not significantly different between groups. We measured Vimentin expression in different groups to investigate the fibroblast-specific expression genes. In iTot cells, the expression of vimentin is decreased compared to the fibroblast cells. Zscan4 and Dux (totipotent stem cell marker and transcribtion factors) also have been investigated. Our data showed cells treated with Aza have higher expression of Zscan4 and Dux versus fibroblasts and ESCs (Fig. 2C).

Dnmt1 and Dnmt3A could mimic the role of Aza in fibroblast cells.
Aza could induce DNA demethylation by targeting DNMTs. In this essence, we hypothesized that the knockdown of these genes could be responsible for induced totipotent stem cells. So, we knock down Dnmt1 and Dnmt3A, which are major Dnmt proteins with methylation activity, and analyze the methylation level of the genome (Fig. 3A,B). Dnmt1 and Dnmt3A co-inhibition results in genome-efficient hypermethylation, activation of MERVL, and the presence of Nanog and Cdx2 positive cells (Fig. 3C-E). These cells were also positive for alkaline phosphatase (Fig. 3F).
Induced totipotent stem cells have developmental potential. After obtaining these results from molecular tests, the functional test is crucial. In this essence, in-vitro differentiation of induced cells into ICM and Trophoblast lineage cells seems necessary. For these purposes, induced cells are cultured in a medium containing LIF and bFGF. The culture of induced cells with LIF results in pluripotent stem cell colonies that are positive for Ssea1 and overexpression of pluripotency-related genes as expected. On the other hand, bFGF-treated cells could lead to trophoblast morphologies that are positive for Elf5 and have upregulated trophoblast-related   www.nature.com/scientificreports/ genes (Fig. 4A,B). In-vivo tests for the evaluation of the full potential of these cells were conducted. At first sight, often-used chimerism testing is supposed to be a good candidate, but due to the large diameter of induced structures, whole structures are injected into pseudopregnant mice. In this regard, we inject GFP-positive iTot cells (Fig. 4C). After injection, ten DPC mothers are dissected (Fig. 4D). The uterus of dissected mice carried sacs (Fig. 4E) containing eight DPC embryo-like structures that were positive for GFP (Fig. 4F).

Discussion
In brief, we report the reprogramming of fibroblast cells to a totipotent state. These cells demonstrate morphology near the normal totipotent stem cells. Most features of these cells have the potential to implant, form, and grow embryo-like structures. However, its genome-scale investigation remains to be elucidated. This research shows the role of demethylation in the initiation of ZGA, induction of totipotency, and its possible role in the initiation of embryo development programming. In this study, Cdx2 and Nanog are chosen as markers to determine embryonic fate. Cdx2 and Nanog are the markers of the trophoblast and epiblast cells and are the main difference between 2 and 4C cells. We determine the expression of related imprinting genes for investigating the reprogramming process. We chose Dppa3 and Dnmt3L, which have great roles in maintaining and establishing the right imprinting pattern, in agreement with a natural process; and we observe the same expression pattern. Apart from the expression of these genes, we investigate the expression of Meg3, which shows its relation to successful chimera engrafting. These data show that our phenotypes have the potential for chimera engraftment. Based on Meg3 results we performed an embryo transfer test which was successful. This data showed that DNA demethylation could induce a totipotent state and validate its role as a determining factor in the development of embryos and zygotes. This reprogramming is based on using one well-known chemical substrate that is labor free and could be done on finally differentiated cells such as fibroblast in comparison with the latest advances which use ESC as the source. Finally, iTot cells could form post-implantation embryos which is an advancement for prior researchers.

Materials and methods
Cell culture. For fibroblast isolation, the ear of the mouse (BALB/c) was minced. The pieces were washed with 75% methanol for two minutes. Next, ethanol was removed by washing with DMEM (Gibco). The next step was to chop the pieces with a surgical blade and trypsinize them at 37 C. The solution was centrifuged and re-suspended in DMEM containing 10% FBS (Invitrogen) and 2% pen-strep (Invitrogen). When the cells reach 70-80% percent confluency, they have been trypsinized and passaged. For treatment, the medium was changed when the cells reached 75-80% confluency, and after two hours, 1.5 µg per milliliter of 5-AzaC (sigma-A2385- www.nature.com/scientificreports/ 100MG) was added. 5-AzaC was dissolved in the culture medium and freshly used every day up to the third day. For the extraction of ES cells, the mice were mated overnight. We let sufficient time pass and then dissected the mice, and 3 DPC embryos were transferred into a plate with a full medium consisting of high glucose DMEM, 103 units per milliliter of LIF (Invitrogen), and 10% of FCS (Invitrogen). Regarding the lineage tracing test, GFP-positive transgenic mice (BALB/c) were provided by the Royan Institute. 103 U/ml and 25 ng/ml of LIF and bFGF (Sigma) were added to iTot cells to differentiate them into pluripotent stem cells and trophoblast lineages.
Alkaline phosphatase assay. To   Embryo transfer. At eight days, cells were trypsinized and relocated to 3.5 cm Petri dishes. After 40 min, the non-attached cells were harvested and relocated to a transfer solution consisting of 10% FBS, 89% DMEM, and 1% antibiotic. Mice were anesthetized with Ketamine. The cells were injected into pseudopregnant NMR mice.
Ethics and animal rights. All experimental protocols were approved by a Shahid Beheshti University institutional licensing committee. All methods were carried out in accordance with Shahid Beheshti University regulations, and ARRIVE guidelines.

Statistical analysis. Statistical analysis has been performed with GraphPad Prism 8. T-test and ANOVA
tests have been performed as it fitted and P value under 0.05 has been considered as significant.

Data availability
All the data generated/analyzed during the study are included in this published article.